Project Summary BRCA1 or BRCA2 mutations increase the risk of several cancers, including breast, ovarian, prostate, and pancreatic cancers. Cells with dysfunctional BRCA1 or BRCA2 gene expression suffer defects in performance of double strand break (DSB) repair mechanisms including homologous recombination (HR) and non- homologous DNA end joining (NHEJ). Cancer treatments exploit this characteristic, the best example of which is the PARP inhibitors (PARPi), which show ?synthetic lethality? with BRCA1 or BRCA2 deficiency due to HR repair defects in BRCA1 or BRCA2-deficient cancer cells. However, resistance to PARPi is a major clinical problem in BRCA-mutated cancers. Unexpectedly, loss of the DNA repair protein 53BP1 renders BRCA1- deficient cancer cells resistant to PARPi and radiation treatment, indicating that loss of 53BP1 expression is at least one scenario by which therapeutic resistance may arise in BRCA-mutated cancers. We and others reported recently that mechanistically, 53BP1 acts as an adaptor protein and controls two downstream sub-pathways, one mediated by PTIP and Artemis and the other mediated by RIF1 and MAD2L2, to coordinate pathologic DSB repair pathway choices in BRCA1-deficient cells. However, the direct upstream regulator(s) of 53BP1 function in DNA repair and cellular response to PARPi and/or irradiation in BRCA-mutated cancers remain unknown. Our recent studies have uncovered several 53BP1-binding proteins that regulates 53BP1 stability, modulates 53BP1 access to DSBs and functions in DNA repair through post-translational modification of 53BP1. Thus, our overall hypothesis is that previously unrecognized 53BP1 repair pathways confer PARPi resistance that can be targeted therapeutically in BRCA-mutated cancers. To address this hypothesis, we propose 3 specific aims: Aim 1: Determine the mechanism underlying TIRR regulation of 53BP1 at DSBs. Aim 2: Determine the role(s) of NUDT16 in 53BP1 regulation and in DNA repair pathways. Aim 3: Reveal the functional significance of HDGFRP3 in damage-induced 53BP1 localization, and DNA repair. Knowledge such as this is essential in identifying those patients with BRCA-mutated cancers who will most benefit from PARPi-based therapies.